Transforming growth factor alpha (TGF-alpha), amphiregulin (AR), heregulin (HRG) and cripto-1 (CR-1) are proteins that are structurally and in some cases functionally related to epidermal growth factor (EGF) in that TGF-alpha and AR can bind to the EGF receptor (c-erb B) whereas HRG binds to c-erb B-3 or c-erb B-4. The present studies have demonstrated that MCF-10A human mammary epithelial cells are mitogenically responsive to exogenous EGF, TGF-alpha or AR and that transformation of these cells with a point-mutated c-Ha-ras protooncogene results in an increase in the expression of endogenous TGF-alpha, AR and HRG whereas erb B-2 transformation of these cells results in an upregulation in AR and HRG expression. Furthermore, overexpression of a human TGF-alpha cDNA in these cells leads to their in vitro transformation. Addition of an anti-EGF receptor blocking antibody inhibits the growth of MCF-10A transformed mammary cells suggesting that an external autocrine loop is operative in these cells. Estrogens can increase the expression of TGF-alpha and AR mRNA and protein in estrogen-responsive human breast cancer cell lines. A recombinant CR-1 protein is able to moderately stimulate the proliferation of mouse and human mammary epithelial cells and to inhibit beta-casein and whey acidic protein expression. CR-1 does not directly bind to the EGF receptor nor does it directly activate the c-erb B-2, c-erb B-3 or c-erb B-4 type 1 receptor tyrosine kinases either singularly or in various heterodimeric pairwise combinations. However, CR-1 can rapidly and transiently enhance the tyrosine phosphorylation of p46 Shc and can activate the MAPK isoform, p42erk2. 125I-CR-1 binds to a 135 kDa tyrosine phosphorylated and membrane-associated protein. Protein and mRNA expression for AR and CR-1 have been detected in approximately 50% to 80% of primary and metastatic human colorectal tumors, whereas only 5% of normal adjacent colon or liver tissue express these genes. Likewise,AR and CR-1 were detected in approximately 80% of primary human breast tumors at a level that exceeded the level found in adjacent normal normal mammary epithelium.